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1.  Genomic and Expression Analysis of Microdissected Inflammatory Breast Cancer 
Purpose
Inflammatory breast cancer (IBC) is a unique clinical entity characterized by rapid onset of erythema and swelling of the breast often without an obvious breast mass. Many studies have examined and compared gene expression between IBC and non-IBC (nIBC), repeatedly finding clusters associated with receptor subtype, but no consistent gene signature associated with IBC has been validated. Here we compared microdissected IBC tumor cells to microdissected nIBC tumor cells matched based on estrogen and HER-2/neu receptor status.
Methods
Gene expression analysis and comparative genomic hybridization were performed. An IBC gene set and genomic set were identified using a training set and validated on the remaining data. The IBC gene set was further tested using data from IBC consortium samples and publically available data.
Results
Receptor driven clusters were identified in IBC; however no IBC-specific gene signature was identified. Fifteen genes were correlated between increased genomic copy number and gene overexpression data. An expression-guided gene set upregulated in the IBC training set clustered the validation set into two clusters independent of receptor subtype but segregated only 75% of samples in each group into IBC or nIBC. In a larger consortium cohort and in published data the gene set failed to optimally enrich for IBC samples. However, this gene set had a high negative predictive value for excluding the diagnosis of IBC in publically available data (100%). An IBC enriched genomic data set accurately identified 10/16 cases in the validation data set.
Conclusions
Even with microdissection, no IBC-specific gene signature distinguishes IBC from nIBC. Using microdissected data, a validated gene set was identified that is associated with IBC tumor cells. IBC comparative genomic hybridization data are presented, but a validated genomic data set that identifies IBC is not demonstrated.
doi:10.1007/s10549-013-2501-6
PMCID: PMC3677826  PMID: 23568481
Inflammatory breast cancer; CGH; array; gene signature
2.  Use of the Cytokinesis-Blocked Micronucleus Assay (CBMN) to Detect Gender Differences and Genetic Instability in a Lung Cancer Case-Control Study 
Background
Although tobacco exposure is the predominant risk factor for lung cancer, other environmental agents are established lung carcinogens. Measuring the genotoxic effect of environmental exposures remains equivocal as increases in morbidity and mortality may be attributed to co-exposures such as smoking.
Methods
We evaluated genetic instability and risk of lung cancer associated with exposure to environmental agents (e.g., exhaust) and smoking among 500 lung cancer cases and 500 controls using the Cytokinesis-Blocked Micronucleus (CBMN) assay. Linear regression was applied to estimate the adjusted means of the CBMN endpoints (micronuclei and nucleoplasmic bridges). Logistic regression analyses were used to estimate lung cancer risk and to control for potential confounding by age, gender, and smoking.
Results
Cases showed significantly higher levels of micronuclei and nucleoplasmic bridges as compared to controls (mean ± SEM=3.54±0.04 vs.1.81 ±0.04 and mean ± SEM=4.26±0.03 vs. 0.99±0.03, respectively; p <0.001) with no differences among participants with or without reported environmental exposure. No differences were observed when stratified by smoking or environmental exposure among cases or controls. A difference in lung cancer risk was observed between non-exposed male and female heavy smokers, although it was not statistically significant (I2=64.9%; P-value for Q statistic=0.09).
Conclusions
Our study confirms that the CBMN assay is an accurate predictor of lung cancer and supports the premise that heavy smoking may have an effect on DNA repair capacity and in turn modulate the risk of lung cancer.
Impact
Identifying factors that increase lung cancer risk may lead to more effective prevention measures.
doi:10.1158/1055-9965.EPI-12-0435
PMCID: PMC3538922  PMID: 23195992
Lung cancer; CBMN assay; DNA damage; gender differences
3.  Using Haplotype Analysis to Elucidate Significant Associations between Genes and Hodgkin Lymphoma 
Leukemia research  2012;36(11):1359-1364.
In this study, we estimated the association between the inferred haplotypes in the inflammation, DNA repair, and folate pathways, and developed risk models for Hodgkin Lymphoma. The study population consisted of 200 Hodgkin Lymphoma cases and 220 controls. A susceptible association was observed on the XPC gene with Haplotype CT (rs2228001 and rs2228000), and a protective association was observed on the IL4R gene with Haplotype TCA (rs1805012, rs1805015, and rs1801275). These results can provide the necessary tools to identify high-risk individuals after validation in large data sets.
doi:10.1016/j.leukres.2012.07.014
PMCID: PMC3449215  PMID: 22902050
Single Nucleotide Polymorphism; Haplotypes; Hodgkin lymphoma; DNA Repair; Inflammation
4.  Insulin-Like Growth Factor Binding Protein-2 Level Is Increased in Blood of Lung Cancer Patients and Associated with Poor Survival 
PLoS ONE  2013;8(9):e74973.
Background
We recently showed that IGFBP2 is overexpressed in primary lung cancer tissues. This study aims to determine whether IGFBP2 is elevated in blood samples of lung cancer patients and whether its level is associated with clinical outcomes.
Methodology/Principal Findings
Plasma IGFBP2 levels were determined blindly by enzyme-linked immunosorbent assay in 80 lung cancer patients and 80 case-matched healthy controls for comparison. We analyzed blood samples for IGFBP2 levels from an additional 84 patients with lung cancer and then tested for associations between blood IGFBP2 levels and clinical parameters in all 164 lung cancer patients. All statistical tests were two-sided and differences with p<0.05 were considered significant. The mean plasma concentration of IGFBP2 in lung cancer patients was significantly higher than that in healthy controls (388.12±261.00 ng/ml vs 219.30±172.84 ng/ml, p<0.001). IGFBP2 was increased in all types of lung cancer, including adenocarcinoma, squamous cell cancer, and small-cell cancer, regardless of patients’ age, sex, or smoking status. IGFBP2 levels were mildly but significantly associated with tumor size and were significantly higher in stage IV than stage I or III disease. A multivariate analysis showed that lung cancer patients whose blood IGFBP2 was higher than 160.9 ng/ml had a poor survival outcome, with a hazard ratio of 8.76 (95% CI 1.12-68.34, p=0.038 after adjustment for tumor size, pathology, and stage). The median survival time for patients with blood IGFBP2 >160.9 ng/ml is 15.1 months; whereas median survival time was 128.2 months for the patients whose blood IGFBP2 was ≤160.9 ng/ml (p =0.0002).
Conclusions/Significance
Blood IGFBP2 is significantly increased in lung cancer patients. A high circulating level of IGFBP2 is significantly associated with poor survival, suggesting that blood IGFBP2 levels could be a prognostic biomarker for lung cancer.
doi:10.1371/journal.pone.0074973
PMCID: PMC3775736  PMID: 24069370
5.  Status of the anaplastic lymphoma kinase (ALK) gene in inflammatory breast carcinoma 
SpringerPlus  2013;2:409.
Background
Although preliminary reports suggest that ALK gene amplification may occur in inflammatory breast cancer (IBC), data are limited. We performed a comprehensive investigation of the status of ALK gene in IBC.
Methods
We used core biopsy (CB) samples from 30 IBC patients for immunohistochemistry (IHC), 25 of these samples for fluorescence in situ hybridization (FISH) of ALK gene rearrangement, 8 for chromosome 2 aneusomy, and 20 microdissected frozen CBs for array comparative genomic hybridization (CGH) and mRNA analysis.
Results
All 30 samples were negative for ALK protein expression by IHC. FISH analysis showed no EML4-ALK gene rearrangement in any samples, although 16 of the 25 samples (64%) contained 3 to 4 extra copies of the ALK gene, and chromosome 2 aneusomy was found in 7 of 8 samples that had extra copies of the ALK gene. Only 3 of the 20 samples showed evidence of mild ALK gene amplification by array CGH. mRNA analysis revealed that mRNA expression of ALK was not significantly higher in these samples compared with samples that showed no evidence of ALK gene amplification in CGH analysis, nor was mRNA expression of ALK significantly different in tumor compared with 5 normal breast samples (P > 0.05, t test).
Conclusion
Our comprehensive evaluation suggests that ALK gene rearrangement did not occur in the IBC patients studied. The significance of our finding of mildly increased copy numbers of the ALK gene resulting from chromosome 2 aneusomy rather than mild amplification of the ALK gene requires further investigation.
doi:10.1186/2193-1801-2-409
PMCID: PMC3797914  PMID: 24156086
6.  Bridging the clinical gaps: genetic, epigenetic and transcriptomic biomarkers for the early detection of lung cancer in the post-National Lung Screening Trial era 
BMC Medicine  2013;11:168.
Lung cancer is the leading cause of cancer death worldwide in part due to our inability to identify which smokers are at highest risk and the lack of effective tools to detect the disease at its earliest and potentially curable stage. Recent results from the National Lung Screening Trial have shown that annual screening of high-risk smokers with low-dose helical computed tomography of the chest can reduce lung cancer mortality. However, molecular biomarkers are needed to identify which current and former smokers would benefit most from annual computed tomography scan screening in order to reduce the costs and morbidity associated with this procedure. Additionally, there is an urgent clinical need to develop biomarkers that can distinguish benign from malignant lesions found on computed tomography of the chest given its very high false positive rate. This review highlights recent genetic, transcriptomic and epigenomic biomarkers that are emerging as tools for the early detection of lung cancer both in the diagnostic and screening setting.
doi:10.1186/1741-7015-11-168
PMCID: PMC3717087  PMID: 23870182
Biomarker; Diagnostics; Early detection; Epigenetics; Genetics; Lung cancer; Screening; Transcriptomics
7.  A Novel Technique for the Assessment of Preoperative Cardiovascular Risk: Reactive Hyperemic Response to Short-Term Exercise 
BioMed Research International  2013;2013:837130.
Background. Perioperative vascular function has been widely studied using noninvasive techniques that measure reactive hyperemia as a surrogate marker of vascular function. However, studies are limited to a static setting with patients tested at rest. We hypothesized that exercise would increase reactive hyperemia as measured by digital thermal monitoring (DTM) in association to patients' cardiometabolic risk. Methods. Thirty patients (58 ± 9 years) scheduled for noncardiac surgery were studied prospectively. Preoperatively, temperature rebound (TR) following upper arm cuff occlusion was measured before and 10 minutes after exercise. Data are presented as means ± SD. Statistical analysis utilized ANOVA and Fisher's exact test, with P values <0.05 regarded as significant. Results. Following exercise, TR-derived parameters increased significantly (absolute: 0.53 ± 0.95 versus 0.04 ± 0.42°C, P=0.04, and % change: 1.78 ± 3.29 versus 0.14 ± 1.27 %, P=0.03). All patients with preoperative cardiac risk factors had a change in TR (after/before exercise, ΔTR) with values falling in the lower two tertiles of the study population (ΔTR <1.1%). Conclusion. Exercise increased the reactive hyperemic response to ischemia. This dynamic response was blunted in patients with cardiac risk factors. The usability of this short-term effect for the preoperative assessment of endothelial function warrants further study.
doi:10.1155/2013/837130
PMCID: PMC3652140  PMID: 23691513
8.  Hodgkin Lymphoma Risk: Role of Genetic Polymorphisms and Gene-Gene Interactions in DNA repair pathways 
Molecular carcinogenesis  2011;50(11):825-834.
DNA repair variants may play a potentially important role in an individual’s susceptibility to developing cancer. Numerous studies have reported the association between genetic single nucleotide polymorphisms (SNPs) in DNA repair genes and different types of hematologic cancers. However, to date, the effects of such SNPs on modulating Hodgkin Lymphoma (HL) risk have not yet been investigated. We hypothesized that gene-gene interaction between candidate genes in Direct Reversal, Nucleotide excision repair (NER), Base excision repair (BER) and Double strand break (DSB) pathways may contribute to susceptibility to HL. To test this hypothesis, we conducted a study on 200 HL cases and 220 controls to assess associations between HL risk and 21 functional SNPs in DNA repair genes. We evaluated potential gene-gene interactions and the association of multiple polymorphisms in a chromosome region using a multi-analytic strategy combining logistic regression, multi-factor dimensionality reduction and classification and regression tree approaches. We observed that, in combination, allelic variants in the XPC Ala499Val, NBN Glu185Gln, XRCC3 Thr241Me, XRCC1 Arg194Trp and XRCC1 399Gln polymorphisms modify the risk for developing HL. Moreover, the cumulative genetic risk score revealed a significant trend where the risk for developing HL increases as the number of adverse alleles in BER and DSB genes increase. These findings suggest that DNA repair variants in BER and DSB pathways may play an important role in the development of HL.
doi:10.1002/mc.20747
PMCID: PMC3131460  PMID: 21374732
9.  Folate Pathway Polymorphisms Predict Deficits in Attention and Processing Speed after Childhood Leukemia Therapy 
Pediatric blood & cancer  2011;57(3):454-460.
Background
Neurocognitive impairment occurs in 20%-40% of childhood acute lymphoblastic leukemia (ALL) survivors, possibly mediated by folate depletion and homocysteine elevation following methotrexate treatment. We evaluated the relationship between folate pathway polymorphisms and neurocognitive impairment after childhood ALL chemotherapy.
Procedure
Seventy-two childhood ALL survivors treated with chemotherapy alone underwent a neurocognitive battery consisting of: Trail Making Tests A (TMTA) and B (TMTB), Grooved Pegboard Test Dominant-Hand and Nondominant-Hand, Digit Span subtest, and Verbal Fluency Test. We performed genotyping for: 10-methylenetetrahydrofolate reductase (MTHFR 677C>T and MTHFR 1298A>C), serine hydroxymethyltransferase (SHMT 1420C>T), methionine synthase (MS 2756 A>G), methionine synthase reductase (MTRR 66A>G), and thymidylate synthase (TSER). Student's two sample t-test and analysis of covariance were used to compare test scores by genotype.
Results
General impairment on the neurocognitive battery was related to MTHFR 1298A>C (p=0.03) and MS 2756A>G (p=0.05). Specifically, survivors with MTHFR 1298AC/CC genotypes scored, on average, 13 points lower on TMTB than those with MTHFR 1298AA genotype (p=0.001). The MS 2756AA genotype was associated with a 12.2 point lower mean TMTA score, compared to MS 2756 AG/GG genotypes (p=0.01). The TSER 2R/3R and 3R/3R genotypes were associated with an 11.4 point lower mean score on TMTB, compared to the TSER 2R/2R genotype (p=0.03). Survivors with >6 folate pathway risk alleles demonstrated a 9.5 point lower mean TMTA score (p=0.06) and 14.5 point lower TMTB score (p=0.002) than survivors with <6 risk alleles.
Conclusions
Folate pathway polymorphisms are associated with deficits in attention and processing speed after childhood ALL therapy.
doi:10.1002/pbc.23162
PMCID: PMC3134130  PMID: 21618410
folate; leukemia; neurocognitive; survivor
10.  Evaluating the effects of genetic variants of DNA repair genes using cytogenetic mutagen sensitivity approaches 
Mutagen sensitivity, measured in short-term cultures of peripheral blood lymphocytes by cytogenetic endpoints, is an indirect measure for DNA repair capacity and has been used for many years as a biomarker for intrinsic susceptibility for cancer. In this article, we briefly give an overview of the different cytogenetic mutagen sensitivity approaches that have been used successfully to evaluate the biological effects of polymorphisms in DNA repair genes based on a current review of the literature and based on the need for biomarkers that would allow the characterization of the biological and functional significance of such polymorphisms. We also address some of the future challenges facing this emerging area of research.
doi:10.3109/1354750X.2011.577237
PMCID: PMC3142279  PMID: 21595606
DNA repair; polymorphisms; haplotype; biomarkers; mutagen sensitivity; cancer; environmental exposure
11.  Gamma-radiation sensitivity and polymorphisms in RAD51L1 modulate glioma risk 
Carcinogenesis  2010;31(10):1762-1769.
Background: DNA strand breaks pose the greatest threat to genomic stability. Genetically determined mutagen sensitivity predisposes individuals to a variety of cancers, including glioma. However, polymorphisms in DNA strand break repair genes that may determine mutagen sensitivity are not well studied in cancer risk, especially in gliomas.
Methods: We correlated genotype data for tag single-nucleotide polymorphisms (tSNPs) of DNA strand break repair genes with a gamma-radiation-induced mutagen sensitivity phenotype [expressed as mean breaks per cell (B/C)] in samples from 426 glioma patients. We also conducted analysis to assess joint and haplotype effects of single-nucleotide polymorphisms (SNPs) on mutagen sensitivity. We further validate our results in an independent external control group totaling 662 subjects.
Results: Of the 392 tSNPs examined, we found that mutagen sensitivity was modified by one tSNP in the EME2 gene and six tSNPs in the RAD51L1 gene (P < 0.01). Among the six RAD51L1 SNPs tested in the validation set, one (RAD51L1 rs2180611) was significantly associated with mutagen sensitivity (P = 0.025). Moreover, we found a significant dose–response relationship between the mutagen sensitivity and the number of adverse tSNP genotypes. Furthermore, haplotype analysis revealed that RAD51L1 haplotypes F-A (zero adverse allele) and F-E (six adverse alleles) exhibited the lowest (0.42) and highest (0.93) mean B/C values, respectively. A similar dose–response relationship also existed between the mutagen sensitivity and the number of adverse haplotypes.
Conclusion: These results suggest that polymorphisms in and haplotypes of the RAD51L1 gene, which is involved in the double-strand break repair pathway, modulate gamma-radiation-induced mutagen sensitivity.
doi:10.1093/carcin/bgq141
PMCID: PMC2981459  PMID: 20610542
12.  Genetically Abnormal Circulating Cells in Lung Cancer Patients: An Antigen Independent Fluorescence in-situ Hybridization Based Case-Control Study 
Purpose
We performed a study to determine if a fluorescence in-situ hybridization (FISH)-based assay using isolated peripheral blood mononuclear cells (PBMCs) with DNA probes targeting specific sites on chromosomes known to have abnormalities in Non Small Cell Lung Cancer (NSCLC) cases could detect circulating genetically abnormal cells (CACs).
Experimental Design
We evaluated 59 NSCLC cases with stage I through IV disease and 24 controls. PBMCs and matched tumors were hybridized with 2 two-color (3p22.1/CEP3 and 10q22.3 [SP-A]/CEP10) and 2 four-color (CEP3, CEP7, CEP17, and 9p21.3 [URO]) and (EGFR, c-MYC, 6p11-q11, and 5p15.2 [LAV]) FISH probes. Percentages of cytogenetically abnormal cells (CACs) in peripheral blood and in matched tumor specimens were quantified using an automated fluorescent scanner. Numbers of CACs were calculated based on the percentage of CACs (defined as PBMCs with genetic abnormalities) per mL of blood and expressed per microliter of blood.
Results
Patients with NSCLC had significantly higher numbers of CACs than did controls. Mean number of CACs ranged from 7.23±1.32/μl for deletions of 10q22.3/CEP10 to 45.52±7.49/μl for deletions of 3p22.1/CEP3. Numbers of CACs with deletions of 3p22.1, 10q22.3, and 9p21.3, and gains of URO, increased significantly from early to advanced stage of disease.
Conclusions
We have developed a sensitive and quantitative antigen-independent FISH-based test for detecting CACs in peripheral blood of patients with NSCLC which showed a significant correlation with the presence of cancer. If this pilot study can be validated in a larger study, CACs may have a role in the management of patients with NSCLC.
doi:10.1158/1078-0432.CCR-09-3358
PMCID: PMC2949278  PMID: 20651054
13.  Genetic evidence linking lung cancer and COPD: a new perspective 
Epidemiological studies indicate that tobacco smoke exposure accounts for nearly 90% of cases of chronic obstructive pulmonary disease (COPD) and lung cancer. However, genetic factors may explain why 10%–30% of smokers develop these complications. This perspective reviews the evidence suggesting that COPD is closely linked to susceptibility to lung cancer and outlines the potential relevance of this observation. Epidemiological studies show that COPD is the single most important risk factor for lung cancer among smokers and predates lung cancer in up to 80% of cases. Genome-wide association studies of lung cancer, lung function, and COPD have identified a number of overlapping “susceptibility” loci. With stringent phenotyping, it has recently been shown that several of these overlapping loci are independently associated with both COPD and lung cancer. These loci implicate genes underlying pulmonary inflammation and apoptotic processes mediated by the bronchial epithelium, and link COPD with lung cancer at a molecular genetic level. It is currently possible to derive risk models for lung cancer that incorporate lung cancer-specific genetic variants, recently identified “COPD-related” genetic variants, and clinical variables. Early studies suggest that single nucleotide polymorphism-based risk stratification of smokers might help better target novel prevention and early diagnostic strategies in lung cancer.
doi:10.2147/TACG.S20083
PMCID: PMC3681182  PMID: 23776371
lung cancer; chronic obstructive pulmonary disease; association study; single nucleotide polymorphism; risk model
14.  A Comprehensive Haplotype Analysis of the XPC Genomic Sequence Reveals a Cluster of Genetic Variants Associated with Sensitivity to Tobacco-Smoke Mutagens 
Toxicological Sciences  2010;115(1):41-50.
The impact of single-nucleotide polymorphisms (SNPs) of the DNA repair gene XPC on DNA repair capacity (DRC) and genotoxicity has not been comprehensively determined. We constructed a comprehensive haplotype map encompassing all common XPC SNPs and evaluated the effect of Bayesian-inferred haplotypes on DNA damage associated with tobacco smoking, using chromosome aberrations (CA) as a biomarker. We also used the mutagen-sensitivity assay, in which mutagen-induced CA in cultured lymphocytes are determined, to evaluate the haplotype effects on DRC. We hypothesized that if certain XPC haplotypes have functional effects, a correlation between these haplotypes and baseline and/or mutagen-induced CA would exist. Using HapMap and single nucleotide polymorphism (dbSNP) databases, we identified 92 SNPs, of which 35 had minor allele frequencies ≥ 0.05. Bayesian inference and subsequent phylogenetic analysis identified 21 unique haplotypes, which segregated into six distinct phylogenetically grouped haplotypes (PGHs A–F). A SNP tagging approach used identified 11 tagSNPs representing these 35 SNPs (r2 = 0.80). We utilized these tagSNPs to genotype a population of smokers matched to nonsmokers (n = 123). Haplotypes for each individual were reconstituted and PGH designations were assigned. Relationships between XPC haplotypes and baseline and/or mutagen-induced CA were then evaluated. We observed significant interaction among smoking and PGH-C (p = 0.046) for baseline CA where baseline CA was 3.5 times higher in smokers compared to nonsmokers. Significant interactions among smoking and PGH-D (p = 0.023) and PGH-F (p = 0.007) for mutagen-induced CA frequencies were also observed. These data indicate that certain XPC haplotypes significantly alter CA and DRC in smokers and, thus, can contribute to cancer risk.
doi:10.1093/toxsci/kfq027
PMCID: PMC2855352  PMID: 20106949
DNA nucleotide excision repair; XPC gene; polymorphism; haplotypes; biomarkers; chromosome; smoking; cancer
15.  Genetic variants in inflammation pathway genes and asthma in glioma susceptibility 
Neuro-Oncology  2010;12(5):444-452.
Single nucleotide polymorphisms (SNPs) in inflammation-related genes have previously been shown to alter risks of developing various cancers. However, the effects of such SNPs on glioma risk remain unclear. We used a multistrategic approach to elucidate the relationship between glioma risk, asthma/allergies, and 23 literature-based functional SNPs in 11 inflammation genes. Genotyping was conducted on 373 histologically confirmed adult glioma patients and 365 cancer-free controls from the Harris County Brain Tumor Study. Deviations from the Hardy–Weinberg equilibrium were assessed using the χ2-test, and Akaike's information criterion was used to determine the best genetic model for each SNP. Odds ratios (ORs) were calculated both for each SNP individually and for grouped analyses, examining the effects of the numbers of adverse alleles on glioma risk in participants with and without asthma. In the single-locus analysis of the 23 examined SNPs, 1 pro-inflammatory and 2 anti-inflammatory gene SNPs were significantly associated with glioma risk (COX2/PTGS2, rs20417 [OR = 1.41]; IL10, rs1800896 [OR = 1.57]; and IL13, rs20541 [OR = 0.39], respectively). When we examined the joint effects of the risk-conferring alleles of these 3 SNPs, we found a significant trend indicating that the risk increases as the number of adverse alleles increase (P = .005). Stratifying by asthma status, we found that this dose–response-like trend of increasing risk is only present among those without asthma/allergies (P < .0001). Our study indicates that polymorphisms in inflammation genes are associated with glioma susceptibility, especially when a history of asthma/allergies is absent.
doi:10.1093/neuonc/nop057
PMCID: PMC2940617  PMID: 20406895
allergy; asthma; glioma; inflammation
16.  Regulatory regions responsive to oxidative stress in the promoter of the human DNA glycosylase gene NEIL2 
Mutagenesis  2009;25(2):171-177.
Reactive oxygen species (ROS) generated endogenously or from exogenous sources produce mutagenic DNA lesions. If not repaired, these lesions could lead to genomic instability and, potentially, to cancer development. NEIL2 (EC 4.2.99.18), a mammalian base excision repair (BER) protein and ortholog of the bacterial Fpg/Nei, excises oxidized DNA lesions from bubble or single-stranded structures, suggesting its involvement in transcription-coupled DNA repair. Perturbation in NEIL2 expression may, therefore, significantly impact BER capacity and promote genomic instability. To characterize the genetic and environmental factors regulating NEIL2 gene expression, we mapped the human NEIL2 transcriptional start site and partially characterized the promoter region of the gene using a luciferase reporter assay. We identified a strong positive regulatory region from nucleotide −206 to +90 and found that expression from this region was contingent on its being isolated from an adjacent strong negative regulatory region located downstream (+49 to +710 bp), suggesting that NEIL2 transcription is influenced by both these regions. We also found that oxidative stress, induced by glucose oxidase treatment, reduced the positive regulatory region expression levels, suggesting that ROS may play a significant role in regulating NEIL2 transcription. In an initial attempt to characterize the underlying mechanisms, we used in silico analysis to identify putative cis-acting binding sites for ROS-responsive transcription factors within this region and then used site-directed mutagenesis to investigate their role. A single-base change in the region encompassing nucleotides −206 to +90 abolished the effect of oxidative stress that was observed in the absence of the mutation. Our study is the first to provide an initial partial characterization of the NEIL2 promoter and opens the door for future research aimed at understanding the role of genetic and environmental factors in regulating NEIL2 expression.
doi:10.1093/mutage/gep058
PMCID: PMC2825343  PMID: 19945985
17.  Detection Of Human Cytomegalovirus In Different Histological Types Of Gliomas 
Acta neuropathologica  2008;116(1):79-86.
The association between human cytomegalovirus (HCMV) infection and glioblastoma has been a source of debate in recent years because of conflicting laboratory reports concerning the presence of the virus in glioma tissue. HCMV is a ubiquitous herpesvirus that exhibits tropism for glial cells and has been shown to transform cells in vitro. Using sensitive immunohistochemical and in situ hybridization methods in 50 glioma samples, we detected HCMV antigen and DNA in 21/21 cases of glioblastoma, 9/12 cases of anaplastic gliomas, and 14/17 cases of low-grade gliomas. Reactivity against the HCMV IE1 antigen (72 kDa) exhibited histology-specific patterns with more nuclear staining for anaplastic and low-grade gliomas, while GBMs showed nuclear and cytoplasmic staining that likely occurs with latent infection. Using IHC, the number of HCMV-positive cells in GBMs was 79% compared to 48% in lower grade tumors. Non-tumor areas of the tissue contained only 4 % and 1% of HCMV-positive cells for GBMs and lower grade tumors, respectively. Hybridization to HCMV DNA in infected cells corresponded to patterns of immunoreactivity. Our findings support previous reports of the presence of HCMV infection in glioma tissues and advocate optimization of laboratory methods for the detection of active HCMV infections. This will allow for detection of low-level latent infections that may play an important role in the initiation and/or promotion of malignant gliomas.
doi:10.1007/s00401-008-0359-1
PMCID: PMC3001277  PMID: 18351367
Glioblastoma; Human cytomegalovirus; immunohistochemistry; in situ hybridization
18.  Polymorphisms in the IL4R gene are associated with better survival in glioma patients 
Purpose
Previous literature provides some evidence that atopic diseases, IgE levels, and inflammatory gene polymorphisms may be associated with risk of glioblastoma. The purpose of this study was to investigate the affects of certain inflammatory gene single nucleotide polymorphisms (SNPs) on patient survival. Malignant gliomas are the most common type of primary brain tumor in adults, however, few prognostic factors have been identified.
Experimental Design
Using 694 incident adult glioma cases identified between 2001 and 2006 in Harris County, Texas, we examined seven SNPs in the interleukin 4, interleukin 13, and interleukin 4-receptor (IL4R) genes. Cox proportional hazards regression was used to examine the association between the SNPs and overall and long-term survival, controlling for age at diagnosis, time between diagnosis and registration, extent of surgical resection, radiation therapy, and chemotherapy.
Results
We found that among high-grade glioma cases, IL4R rs1805016 (TT vs. GT/GG) was significantly protective against mortality over time (HR: 0.59; 95% CI: 0.40–0.88). The IL4R rs1805016 and rs1805015 TT genotypes were both found to be significantly associated with survival beyond one year among high-grade glioma patients (HR: 0.44; 95% CI: 0.27–0.73 and HR: 0.63; 95% CI: 0.44–0.91, respectively). Furthermore, the IL4R haplotype analysis showed that SNPs in the IL4R gene may be interacting together to affect long-term survival among high-grade glioma cases.
Conclusions
These findings indicate that polymorphisms in inflammation pathway genes may play an important role in glioma survival. Further research on the effects of these polymorphisms on glioma prognosis is warranted.
doi:10.1158/1078-0432.CCR-07-4681
PMCID: PMC2995250  PMID: 18927306
glioma; survival; IL-4 receptor; inflammation
19.  Aggregation of Cancer in First-degree Relatives of Patients with Glioma 
Background
Previous studies have been inconclusive in estimating the risk of different cancer sites among close relatives of glioma patients; however, malignant melanoma has been consistently described.
Methods
We obtained family history information from 1,476 glioma patients under age 75 who registered at M.D. Anderson Cancer Center between June 1992 and June 2006. The number of observed cancers (N=1,001) among 8,746 first-degree relatives (FDRs) were compared to the number expected from age-, sex-, and calendar-year specific rates from the Surveillance, Epidemiology, and End Results Program using standardized incidence ratios (SIRs).
Results
The overall SIR for any cancer was 1.21 (95% CI; 1.14 – 1.29). Among FDRs under 45 years, the overall SIR was 5.08 and for relatives >45 the overall SIR was 0.95. The SIRs were significantly elevated for brain tumors (2.14), melanoma (2.02), and sarcoma (3.83). We observed an excess of pancreatic cancer which was significantly higher only among mothers.
Conclusion
We observed an overall 21% increase in cancer among the FDRs of glioma patients, including excess cases of brain tumors and melanoma which could point to similar genetic contributions to these two malignancies. A large international linkage study is underway to examine potential genomic regions important for familial glioma.
doi:10.1158/1055-9965.EPI-07-0576
PMCID: PMC2995005  PMID: 18006942
aggregation; cancer; glioma; first-degree relatives
20.  Rapid method for determination of DNA repair capacity in human peripheral blood lymphocytes amongst smokers 
BMC Cancer  2010;10:439.
Background
DNA repair capacity is an important determinant of susceptibility to cancer. The hOGG1 enzyme is crucial for repairing the 8-oxoguanine lesion that occurs either as a byproduct of oxidative metabolism or as a result of exogenous sources such as exposure to cigarette smoke. It has been previously reported that smokers with low hOGG1 activity had significantly higher risk of developing lung cancer as compared to smokers with high hOGG1 activity.
Methods
In the current study we elucidate the association between plasma levels of 8-OHdG and the OGG1 repair capacity. We used the commercially available 8-OHdG ELISA (enzyme-linked immunosorbent assay), the Comet assay/FLARE hOGG1 (Fragment Length Analysis by Repair Enzymes) assay for quantification of the levels of 8-OHdG and measured the constitutive, induced and unrepaired residual damage, respectively. We compared the DNA repair capacity in peripheral blood lymphocytes following H2O2 exposure in 30 lung cancer patients, 30 non-, 30 former and 30 current smoker controls matched by age and gender.
Results
Our results show that lung cancer cases and current smoker controls have similar levels of 8-OHdG lesions that are significantly higher compared to the non-smokers controls. However, lung cancer cases showed significantly poorer repair capacity compared to all controls tested, including the current smokers controls. After adjustment for age, gender and family history of smoking-related cancer using linear regression, we observed a 5-fold increase in risk of lung cancer associated with high levels of residual damage/reduced repair capacity. Reduced OGG1 activity could be expected to be a risk factor in other smoking-related cancers.
Conclusion
Our study shows that the Comet/FLARE assay is a relatively rapid and useful method for determination of DNA repair capacity. Using this assay we could identify individuals with high levels of residual damage and hence poor repair capacity who would be good candidates for intensive follow-up and screening.
doi:10.1186/1471-2407-10-439
PMCID: PMC2933626  PMID: 20718982
21.  Association and Interactions between DNA Repair Gene Polymorphisms and Adult Glioma 
It is generally accepted that glioma develops through accumulation of genetic alterations. We hypothesized that polymorphisms of candidate genes involved in the DNA repair pathways may contribute to susceptibility to glioma. To address this possibility, we conducted a study of 373 Caucasian glioma cases and 365 cancer-free Caucasian controls to assess associations between glioma risk and 18 functional SNPs in DNA repair genes. We evaluated potential gene-gene and gene-environment interactions using a multi-analytic strategy combining logistic regression, multifactor dimensionality reduction (MDR), and classification and regression tree (CART) approaches. In the single-locus analysis, six SNPs (ERCC1 3’ UTR, XRCC1 R399Q, APEX1 E148D, PARP1 A762V, MGMT F84L, and LIG1 5’UTR) showed a significant association with glioma risk. In the analysis of cumulative genetic risk of multiple SNPs, a significant gene-dosage effect was found for increased glioma risk with increasing numbers of adverse genotypes involving the above-mentioned six SNPs (P trend = 0.0004). Further, both the MDR and CART analyses identified MGMT F84L as the predominant risk factor for glioma, and revealed strong interactions among ionizing radiation (IR) exposure, PARP1 A762V, MGMT F84L and APEX1 E148D. Interestingly, the risk for glioma was dramatically increased in IR exposure individuals who had the wild-type genotypes of both MGMT F84L and PARP1 A762V [adjusted odds ratios (OR), 5.95; 95% confidence intervals (CI), 2.21–16.65]. Taken together, these results suggest that polymorphisms in DNA repair genes may act individually or together to contribute to glioma risk.
doi:10.1158/1055-9965.EPI-08-0632
PMCID: PMC2917049  PMID: 19124499
22.  Genetic Polymorphisms in DNA Repair Genes as Modulators of Hodgkin Disease Risk 
Cancer  2009;115(8):1651-1659.
BACKGROUND
Although the pathogenesis of Hodgkin disease (HD) remains unknown, the results of epidemiologic studies suggest that heritable factors are important in terms of susceptibility. Polymorphisms in DNA repair genes may contribute to individual susceptibility for development of different cancers. However, to the authors’ knowledge, few studies to date have investigated the role of such polymorphisms as risk factors for development of HD.
METHODS
The authors evaluated the relation between polymorphisms in 3 nucleotide excision repair pathway genes (XPD [Lys751Gln], XPC [Lys939Gln], and XPG [Asp1104His]), the base excision repair XRCC1 (Arg399Gln), and double-strand break repair XRCC3 (Thr241Met) in a population of 200 HD cases and 220 matched controls. Variants were investigated independently and in combination; odd ratios (OR) were calculated.
RESULTS
A positive association was found for XRCC1 gene polymorphism Arg399Gln (OR, 1.77; 95% confidence interval [95% CI], 1.16−2.71) and risk of HD. The combined analysis demonstrated that XRCC1/XRCC3 and XRCC1/XPC polymorphisms were associated with a significant increase in HD risk. XRCC1 Arg/Arg and XRCC3 Thr/Met genotypes combined were associated with an OR of 2.38 (95% CI, 1.24−4.55). The XRCC1 Arg/Gln and XRCC3 Thr/Thr, Thr/Met, and Met/Met genotypes had ORs of 1.88 (95% CI, 1.02−4.10), 1.97 (95% CI, 1.05−3.73), and 4.13 (95% CI, 1.50−11.33), respectively. XRCC1 Gln/Gln and XRCC3 Thr/Thr variant led to a significant increase in risk, with ORs of 3.00 (95% CI, 1.15−7.80). Similarly, XRCC1 Arg/Gln together with XPC Lys/Lys was found to significantly increase the risk of HD (OR, 2.14; 95% CI, 1.09−4.23).
CONCLUSIONS
These data suggest that genetic polymorphisms in DNA repair genes may modify the risk of HD, especially when interactions between the pathways are considered.
doi:10.1002/cncr.24205
PMCID: PMC2854485  PMID: 19280628
DNA repair; Hodgkin disease; susceptibility; genetic polymorphisms
23.  Cytokinesis-Blocked Micronucleus Cytome Assay Biomarkers Identify Lung Cancer Cases Amongst Smokers 
The multi-endpoint cytokinesis-blocked micronucleus assay is used for assessing chromosome aberrations. We have recently reported that this assay is extremely sensitive to genetic damage caused by the tobacco-specific nitrosamine 4-(methylnitrosamino)-1-(3-pyr-idyl)-1-butanone (NNK) and that the binucleated cells with micronuclei, nucleoplasmic bridges, and nuclear buds in lymphocytes (chromosome damage endpoints measured by the assay) are strong predictors of lung cancer risk. In the current study, we refined our analysis to include toxicity endpoints (micronuclei in mononucleated cells, apoptosis, necrosis, and nuclear division index) to investigate the benefit of including these variables on improving the predictive value of the assay. Baseline and NNK-induced micronuclei in mononucleated cells were significantly higher in patients (n = 139) than controls (n = 130; P < 0.001). Baseline apoptosis was higher among cases; however, the controls showed a significant higher fold increase in NNK-induced apoptosis compared with baseline (P < 0.001). Principal components analysis was used to derive a summary measure for all endpoints and calculate the positive predictive value (PPV) and negative predictive value (NPV) for disease status. First principal component for NNK-induced chromosome damage endpoints (binucleated cells with micronuclei, nucleoplasmic bridges, and nuclear buds) had an area under the curve = 97.9 (95% confidence interval, 95.9-99.0), PPV = 94.8, and NPV = 92.6. The discriminatory power improved when micronuclei in mononucleated cells were included: area under the curve = 99.1 (95% confidence interval, 97.9- 100.0), PPV = 98.7 and NPV = 95.6. The simplicity, rapidity, and sensitivity of the assay together with potential for automation make it a valuable tool for screening and prioritizing potential cases for intensive screening.
doi:10.1158/1055-9965.EPI-07-2827
PMCID: PMC2854407  PMID: 18483333
24.  Modulation of Radiation-Induced Genetic Damage by HCMV in Peripheral Blood Lymphocytes from a Brain Tumor Case-Control Study 
Cancers  2010;2(2):420-435.
Human cytomegalovirus (HCMV) infection occurs early in life and viral persistence remains through life. An association between HCMV infection and malignant gliomas has been reported, suggesting that HCMV may play a role in glioma pathogenesis and could facilitate an accrual of genotoxic damage in the presence of γ-radiation; an established risk factor for gliomas. We tested the hypothesis that HCMV infection modifies the sensitivity of cells to γ-radiation-induced genetic damage. We used peripheral blood lymphocytes (PBLs) from 110 glioma patients and 100 controls to measure the level of chromosome damage and cell death. We evaluated baseline, HCMV-, γ-radiation and HCMV + γ-radiation induced genetic instability with the comprehensive Cytokinesis-Blocked Micronucleus Cytome (CBMN-CYT). HCMV, similar to radiation, induced a significant increase in aberration frequency among cases and controls. PBLs infected with HCMV prior to challenge with γ-radiation led to a significant increase in aberrations as compared to baseline, γ-radiation and HCMV alone. With regards to apoptosis, glioma cases showed a lower percentage of induction following in vitro exposure to γ-radiation and HCMV infection as compared to controls. This strongly suggests that, HCMV infection enhances the sensitivity of PBLs to γ-radiation-induced genetic damage possibly through an increase in chromosome damage and decrease in apoptosis.
doi:10.3390/cancers2020420
PMCID: PMC3835085  PMID: 24281077
HCMV; brain tumors; CBMN-CYT assay; chromosome aberrations

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